The control of δ-crystallin gene expression during lens cell development: Dissociation of cell elongation,cell division, δ-crystallin synthesis,and δ-crystallin mRNA accumulation

Previous studies have shown that freshly explanted 6-day-old embryonic chick lens epithelial cells elongate, differentially increase their synthesis of δ-crystallin, and accumulate δ-crystallin mRNA when cultured with fetal calf serum; in contrast, precultured serum-starved 6-day-old and freshly explanted 19-day-old embryonic epithelial cells divide when treated with fetal calf serum. We have explored whether the stimulation of δ-crystallin gene expression (as measured by δ-crystallin synthesis and δ-crystallin mRNA accumulation) is affected by inhibiting lens cell elongation with colchicine, and whether δ-crystallin gene expression is increased in lens epithelial cells stimulated to divide by treatment with fetal calf serum, as it is in those stimulated to elongate by treatment with serum. Three new findings were made in this study. First, the stimulation of δ-crystallin gene expression does not require elongation of the cultured lens cells. Second, a decreased proportion of δ-crystallin synthesis is observed in lens epithelial cells during normal development and during serum starvation; in neither case is this decrease associated with a reduction in the number of δ-crystallin mRNA sequences per cell. Finally, serum stimulation of lens cell division does not increase the proportion of δ-crystallin synthesis, but can promote the accumulation of δ-crystallin mRNA. Thus, the relative proportion of δ-crystallin synthesized during chick lens development is not solely a function of the number of δ-crystallin mRNA sequences in the lens cells.     

Developmental regulation of the chicken δ1-crystallin gene: analysis by transgenesis and gene dissection

We previously reviewed what we had learned about the regulation of the δ1-crystallin gene through experiments using gene transfer techniques [Kondoh et al. (1986) Cell Differ. 19, 151–160]. It was concluded then that regulatory genetic elements for the lens-specific expression are associated with the δ1-crystallin gene, and that these chicken elements properly function in mammalian cells. In the last couple of years, we have made significant progress in the understanding of lens-specific δ-crystallin expression. This is owing to success in transgenesis of mouse with the δ1-crystallin gene and in functional dissection of the gene which led us to the discovery of an intragenic enhancer as the major determinant for lens-specific expression. In this article, we summarize these recent advances.     

Enzymic synthesis of argininosuccinate catalyzed by δ-crystallin

Summary A fast and practical procedure has been developed for the synthesis of argininosuccinate using immobilized duck lens -crystallin as catalyst.     

The γ-crystallin gene families: Sequence and evolutionary patterns

Summary The -crystallin proteins consist of two topologically equivalent domains, each built up out of two similar motifs. They are encoded by a gene family, which already contained five members before the divergence of rodents and primates. A further gene duplication took place in each lineage. To analyze the pattern of evolution within this gene family, the coding sequences of six human genes, six rat genes, and four mouse genes were compared. Between species, a uniform rate of evolution of all regions of the protein is seen. The ratio of synonymous to nonsynonymous substitution in the human/rat or human/mouse comparison is much lower than the ratio when rat and mouse are compared indicating that the -crystallin proteins are better conserved in the rodent lineage. Within species, the regions encoding the two external motifs I and III of the protein show a greater extent of nonsynonymous substitution than the regions encoding the two internal protein motifs II and IV. The low extent of synonymous substitution between the second exons (encoding motifs I and II) of the rat -crystallin genes suggests the frequent occurrence of gene conversion. In contrast, a high extent of synonymous substitution is found in exon 3 (encoding motifs III and IV) of the rat genes. The same phenomenon is seen within the human gene family. The frequencies of occurrence of the various dinucleotides deviate less from those predicted from the frequencies of occurrence of each individual nucleotide in the second exons than in the third exons. The sequences of the third exons are significantly depleted in CpG, ApA, and GpT and enriched in CpT and GpA.     

Degradation of δ-crystallin mRNA in the lens fiber cells of the chicken

δ-Crystallin is the principal protein synthesized in the embryonic chicken lens. After hatching δ-crystallin synthesis decreases and eventually ceases. We have determined when the δ-crystallin messenger RNA (mRNA) disappears from the lens fiber cells during the first year of age by cell-free translation of lens RNA in a reticulocyte lysate, RNA blot (Northern) hybridization, and in situ hybridization. The hybridization was performed with a nick-translated, cloned δ-crystallin cDNA (pδCr2). δ-Crystallin mRNA was present in the lens until 3 months of age and disappeared between the third and fifth month after hatching. The in situ hybridization experiments indicated that the δ-crystallin mRNA was present throughout the lens fiber mass until 1 month after hatching and was greatly reduced in the cortical fiber cells thereafter. In contrast to earlier stages, then, the cortical fiber cells differentiating at the lens equator after about 1 month of age do not accumulate δ-crystallin mRNA. The data also indicate that the maximal half-life of functional δ-crystallin mRNA in the posthatched chicken lens is about 2 months.     

Genetic analysis of transgenome structure and size of chromosome-mediated gene transfer lines

The TK-selected chromosome-mediate gene transferlines were analysed using DNA dot blot method,G-11banding and in situ hybridization.The results showedthat CMGT can provide a wide variety of intermediatesize of the transgenome from greater than 80,000kb toless than 2,000kb.Some of transfectants are intergratedinto mouse chromosome which can be detected by G-11banding and in situ hybridization     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic δ-crystallin

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens.The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of δ-crystallin, since δ-crystallin peptides could be identified in tryptic pepetide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable δ-crystallin peptides, although it is possible that modified δ-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added δ-[³⁵S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of δ-crystallin in lens membrane preparations.     

Characterization of the MHC class II ��-chain gene in ducks

In humans, classical MHC class II molecules include DQ, DR, and DP, which are similar in structure but consist of distinct α- and β-chains. The genes encoding these molecules are all located in the MHC class II gene region. In non-mammalian vertebrates such as chickens, only a single class II α-chain gene corresponding to the human DRA has been identified. Here, we report a characterization of the duck MHC class II α-chain (Anpl-DRA) encoding gene, which contains four exons encoding a typical signal peptide, a peptide-binding α1 domain, an immunoglobulin-like α2 domain, and Tm/Cyt, respectively. This gene is present in the duck genome as a single copy and is highly expressed in the spleen. Sequencing of cDNA and genomic DNA of the Anpl-DRA of different duck individuals/strains revealed low levels of genetic polymorphism, especially in the same strain, although most duck individuals have two different alleles. Otherwise, we found that the duck gene is located next to class II β genes, which is the same as in humans but different from the situation in chickens.     

Preliminary X-ray crystallographic study of the turkey lens protein, δ-crystallin

The structural protein, δ-crystallin, has been purified and crystallized from adult turkey lens. The crystals are orthorhombic and normally belong to space group P2₁2₁2 with unit cell dimensions $\text{a = 99.9(2)}\text{A}\text{?}\text{, b = 133.4(3)}\text{A}\text{?}\text{and}\text{c = 69.1(2)}\text{A}\text{?}$. This corresponds to two molecules of molecular weight approximately 200,000 per unit cell. A second crystal form has also been found in which b and c increase to 135.4(3) Å and 140.0(3) Å. respectively, indicating four molecules per unit cell.     

Subunit structure and gene control of mouse NADP—malate dehydrogenase

The NADP-dependent malate dehydrogenase (MDH) in the supernatant fraction of mouse tissues is known to occur in two allelic forms which are electrophoretically distinguishable; each produces a single band in starch gel. We have investigated the subunit structure and synthesis of NADP—MDH through electrophoretic patterns obtained from several experimental sources. (1) Heterozygotes containing both alleles yield a five-banded pattern. The bands are in an approximate frequency of 1:4:6:4:1; the two extremes correspond to the pure types and the three intermediates are presumably hybrid enzymes. The NADP—MDH molecule therefore appears to be a tetramer. (2) In muscle heterokaryons of allophenic mice (with homozygous nuclei of each genotype within a common cytoplasm), hybrid enzymes are formed; they are not formed in other allophenic tissues. Therefore the gene at this locus codes only for monomeric subunits and the tetramer is assembled in a second step in the cytoplasm. Also, both genes must function in a nucleus when the locus is active (e.g., in F₁ uninucleated cells). (3) Dissociation in vitro of mixtures of both pure types of enzymes, followed by reassociation among fragments, leads to a three-banded pattern, even after repeated cycles. Thus the tetramer must cleave in a fixed plane, to form dimers, which reassociate, rather than in a random fashion to form monomers. The most likely interpretation is that mouse NADP—MDH is an example of the type of tetramer postulated by Monod et al. (1965) and termed isologous. The dimers are held symmetrically in the tetrameric conformation by relatively weak forces; the monomeric subunits comprising the dimer are held together by stronger forces.These investigations were supported by U.S.P.H.S. grants No. HD-01646, CA-06927, and FR-05539, and by an appropriation from the Commonwealth of Pennsylvania.     

The RAD6 gene of yeast: A link between DNA repair,chromosome structure and protein degradation?

Summary Among the DNA repair mutants of the yeastSaccharomyces cerevisiae, the rad6 mutants are characterized by a highly pleiotropic phenotype. Most remarkably, these mutants are sensitive towards a variety of DNA-damaging agents and deficient in mutation induction. The RAD6 gene has been cloned and most recently, ubiquitin-conjugating activity of the Rad6 protein has been demonstrated. In this brief review, the properties of rad6 mutants are discussed in the light of these new findings. The available data hint at a connection between DNA repair, mutagenesis, chromosome structure and protein degradation.     

Three-dimensional structure of α-crystallin domain dimers of human small heat shock proteins HSPB1 and HSPB6

Small heat shock proteins (sHSPs) are a family of evolutionary conserved ATP-independent chaperones. These proteins share a common architecture defined by a signature α-crystallin domain (ACD) flanked by highly variable N- and C-terminal extensions. The ACD, which has an immunoglobulin-like fold, plays an important role in sHSP assembly. This domain mediates dimer formation of individual protomers, which then may assemble into larger oligomers. In vertebrate sHSPs, the dimer interface is formed by the symmetrical antiparallel pairing of two β-strands (β7), generating an extended β-sheet on one face of the ACD dimer. Recent structural studies of isolated ACDs from a number of vertebrate sHSPs suggest a variability in the register of the β7/β7 strand interface, which may, in part, give rise to the polydispersity often associated with the full-length proteins. To further analyze the structure of ACD dimers, we have employed a combination of X-ray crystallography and solution small-angle X-ray scattering (SAXS) to study the ACD-containing fragments of human HSPB1 (HSP27) and HSPB6 (HSP20). Unexpectedly, the obtained crystal structure of the HSPB1 fragment does not reveal the typical β7/β7 dimers but, rather, hexamers formed by an asymmetric contact between the β4 and the β7 strands from adjacent ACDs. Nevertheless, in solution, both ACDs form stable dimers via the symmetric antiparallel interaction of β7 strands. Using SAXS, we show that it is possible to discriminate between different putative registers of the β7/β7 interface, with the results indicating that, under physiological conditions, there is only a single register of the strands for both proteins.     

In vitro interactions of histones and α-crystallin

The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed in vitro analyses to determine whether α-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone–α-crystallin binding with a K_d of 4 × 10^?7 M, and the thermodynamic parameters suggested that the interaction was both entropy and enthalpy driven. Size-exclusion chromatography further showed that histone–α-crystallin complexes are water soluble but become water insoluble as the concentration of histones is increased. Right-angle light scattering measurements of the water-soluble fractions of histone–α-crystallin mixtures showed a decrease in the oligomeric molecular weight of α-crystallin, indicating that histones alter the oligomerization of α-crystallin. Taken together, these findings reveal for the first time that histones interact with and affect the solubility and aggregation of α-crystallin, indicating that the interaction between α-crystallin and histones in the lens is functionally important.     

Genetic analysis of transgenome structure and size of chromosome—mediated gene transfer lines

The TK-selected chromosome-mediate gene transfer lines were analysed using DNA dot blot method G-11 banding and in situ hybridization.The results showed that CMGT can provide a wide variety of intermediate size of the transgenome from greater than 80,000kb to less than 2,000kb,Some of transfectants are intergrated into mouse chromosome which can be detected by G-11 banding and in situ hybridization.     

Regulation of adipocyte differentiation and gene expression-crosstalk between TGFβ and wnt signaling pathways

Obesity results in reduced differentiation potential of adipocytes leading to adipose tissue insulin resistance. Elevated proinflammatory cytokines from adipose tissue in obesity, such as TNFα have been implicated in the reduced adipocyte differentiation. Other mediators of reduced adipocyte differentiation include TGFβ and wnt proteins. Although some overlap exists in the signaling cascades of the wnt and TGFβ pathways it is unknown if TGFβ or wnt proteins reciprocally induce the expression of each other to maximize their biological effects in adipocytes. Therefore, we investigated the possible involvement of TGFβ signaling in wnt induced gene expression and vice versa in 3T3-L1 adipocyte. Effect of TGFβ and Wnt pathways on differentiation was studied in preadipocytes induced to differentiate in the presence of Wnt3a or TGFβ1 and their inhibitors (FZ8-CRD and SB431542, respectively). Regulation of intracellular signaling and gene expression was also studied in mature adipocytes. Our results show that both TGFβ1 and Wnt3a lead to increased accumulation of β-catenin, phosphorylation of AKT and p44/42 MAPK. However, differences were found in the pattern of gene expression induced by the two proteins suggesting that distinct, but complex, signaling pathways are activated by TGFβ and wnt proteins to independently regulate adipocyte function.